Biology subjects

Miao, L.

Publications and source records attributed to Miao, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Synergistic integration of inducible RNA switches enhances the manipulation of vector expression

Precise manipulation of gene expression is pivotal for gene function studies and the optimization of gene therapy. RNA-based gene switches are attractive tools due to their robust tunability by FDA-approved small molecules, the absence of exogenous immunogenic proteins, and the small size for gene delivery vectors such as adeno-associated virus (AAV). However, existing RNA switches only target a single step of gene expression such as transcription or RNA splicing, exhibiting intrinsic limitations in gene regulation. To overcome this issue, this study integrated the aptamer-based polyA regulator (pA), the drug-elicitable alternative splicing module (DreAM) and an engineered translation modulator with conditional upstream open reading frames (uORFs) to construct the DreAM-plus RNA switch. The pA-DreAM concatenation led to 1.5[~]5.0-fold and 1.2[~]4.4-fold increase of inducible fold changes than pA and DreAM, respectively. The uORF module further enhanced the switching performance by 1.4[~]6.3-fold. DreAM-plus-mediated transient transgene expression demonstrated a temporal resolution of about 24 hours and high tissue specificity to liver or heart. Critically, DreAM-plus achieved transient expression of an array of gene editors (SpCas9, SaCas9, Un1Cas12f1, OsCas12f1, AcCas12n, IsDra2 TnpB etc.) that significantly mitigated off-target effects by 1.4[~]2.8 folds in plasmids, lentivirus and AAV. In a new mouse model with lipid-nanoparticle-delivered pre-existing immunity, DreAM-plus attenuated AAV-delivered Cas-specific CD8 T cell immune toxicity in the liver and the heart. Therefore, multiple RNA switches could be synergistically integrated to build more sophisticated genetic cassettes for enhanced manipulation of gene expression.

bioengineering↗

bioRxiv2026

Lamin A/C directs nucleosome-scale chromatin remodeling to define early lineage segregation in mammals

Chromatin organization underlies gene regulation and cell fate specification, yet how nucleosome-scale chromatin structure contributes to lineage segregation during early development remains unknown. Here, we resolved chromatin ultrastructure during the first lineage decision in mouse and human, which forms the pluripotent inner cell mass (ICM) and trophectoderm (TE). To achieve this, we developed dual-tilt chromatin electron tomography (2T-ChromEMT) that allows multiscale visualization of chromatin architecture. Our analysis reveals that TE cells of both species display denser chromatin with nucleosome aggregation at the nuclear periphery. We show upregulation of the nuclear matrix protein Lamin A/C within the TE lineage across mouse, human, and opossum embryos, indicating that its regulatory role is conserved across eutherian and marsupial species. Loss of Lamin A/C reduces heterochromatin at the nuclear lamina in TE cells, reactivates pluripotency genes, and impairs mouse blastocyst expansion and human blastoid formation. These findings define the nucleosome-resolution chromatin signatures of early mammalian lineages and establish Lamin A/C-mediated chromatin organization as a conserved mechanism in the exit from pluripotency and maintenance of trophectoderm identity.

developmental biology↗

bioRxiv2025

A stress-dependent TRIM28-ALKBH2 feedback loop modulates chemotherapy response

Chemoresistance to DNA-damaging agents, including platinum and alkylating compounds, limits treatment efficacy in non-small cell lung cancer (NSCLC) and is frequently associated with elevated DNA repair activity. Here, we identify a stress-responsive feedback loop between the E3 ligase TRIM28 and the demethylase ALKBH2. TRIM28 binds ALKBH2 and promotes its K48-linked polyubiquitination and proteasomal degradation, whereas ALKBH2 enhances TRIM28 transcription and protein levels, forming a feedback loop. Notably, alkylation stress induces a biphasic response: acute MMS exposure enhances TRIM28-ALKBH2 association and accelerates ALKBH2 degradation, whereas prolonged exposure promotes TRIM28 degradation, leading to ALKBH2 stabilization and transcriptional upregulation. Clinically, ALKBH2 is frequently elevated in lung adenocarcinoma and is associated with worse survival, whereas TRIM28 exhibits prognostic value specifically in chemotherapy-treated patients. Functionally, MMS-cisplatin co-treatment increased DNA damage and reduced clonogenic survival by counteracting ALKBH2-dependent alkylation tolerance, linking the TRIM28-ALKBH2 loop to chemoresistance in NSCLC.

biochemistry↗

bioRxiv2025

Bioinformatic analysis and experimental validation of ursolic acid's effects on Staphylococcus aureus-induced osteomyelitis

Osteomyelitis (OM), particularly methicillin-resistant Staphylococcus aureus (MRSA)-induced OM, remains a serious clinical challenge due to complex pathogenesis and rising antibiotic resistance. This study aimed to elucidate the therapeutic mechanism of ursolic acid (UA) against MRSA- induced OM using integrated network pharmacology, bioinformatics, and experimental validation. Through target prediction and multi-database mining, we identified 57 common targets of UA and OM. ML algorithms, including Least Absolute Shrinkage and Selection Operator (LASSO) and random forest, pinpointed three core genes: Neutrophil Elastase (ELANE), Lactoferrin (LTF), and S100A12. Enrichment analyses revealed significant involvement in neutrophil extracellular trap (NET) formation, inflammatory response, and immune regulation. Molecular docking and dynamics simulations confirmed stable binding between UA and ELANE. In vitro, UA exhibited antibacterial activity against clinical MRSA isolates (MIC = 16-64 g/mL) and showed synergistic effects with Penicillin (FICI = 0.125-0.5). These results demonstrate that UA combats OM through immunomodulation, anti-inflammatory actions, and direct antibacterial activity, providing a foundation for its use as an adjuvant therapy against MRSA-related OM.

microbiology↗

bioRxiv2025

Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

Transforming growth factor {beta} (TGF{beta}) is a secreted growth factor that is sequestered to the extracellular matrix (ECM) as a latent complex. In adult disease TGF{beta} release in the heart transforms fibroblasts into a differentiated state that synthesizes more ECM. However, it is not known how TGF{beta} functions in the early developing heart to impact resident fibroblasts. Here, we observe that deletion of the Tgfb1, Tgfb2, and Tgfb3 genes (TGF{beta} ligands) from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte-deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness. Gene expression profiling of hearts from myocyte-specific Tgfb1/2/3 deleted mice reveal defects in both cardiomyocyte and fibroblast maturation with ectopic expression of multiple skeletal muscle-specific genes beginning at embryonic day 17.5 and progressing with age. However, cardiomyocyte-specific deletion of TGF{beta} receptors I/II encoding genes (Tgfbr1/2) or Smad2/3 encoding genes (Smad2/3) do not recapitulate this phenotype suggesting that TGF{beta} directly programs early heart fibroblast development that in turn specifies cardiomyocyte maturation. Importantly, Col1a2-/-;Col6a2-/- mice with defective cardiac ECM stiffness, mice lacking cardiomyocyte Itgb1 with reduced ECM load sensing, and Tcf21-/- embryos at E17.5 lacking cardiac fibroblasts each fail to generate the same pathologic ECM program with ectopic cardiomyocyte differentiation observed with Tgfb1/2/3 myocyte-specific deletion. These and additional results indicate that TGF{beta} generated by cardiomyocytes in the embryonic heart mediates fibroblast differentiation that co-evolves the ECM environment that in turn programs cardiomyocyte maturation to establish their identity.

developmental biology↗

bioRxiv2024

A General Strategy to Endow Dyes with Genic Fluorescence for Wash-Free Imaging of Proteins in Living Cells

Fluorescent dyes are the main fluorophore for protein labeling and fluorescent imaging in living cells. However, due to the inevitable nonspecific binding of the dye to non-targets within the cell, background signals are generated, which severely affect the imaging quality. Here, we endow the dye with genic fluorescence by introducing a fluorescent proteins FRET pair, such that only the dye bound to the target protein emits fluorescence ({lambda}ex = FPs, {lambda}em = Dyes), while nonspecifically bound dye remains non-fluorescent. This significantly improves the signal-to-noise ratio (SNR) in wash-free imaging. This strategy is achieved by fusing the Halo-tag to fluorescent proteins (sfGFP, mCherry) that can generate FRET with Halo dyes O-Rho and Si-Rho. The FPs serve as the donor, and the FRET mechanism imparts genic fluorescence property to O-Rho and Si-Rho. Since the excitation wavelength of the donor ({lambda}ex = FPs) cannot excite the unspecifically dye fluorescence, background fluorescence interference is reduced. We further improved the SNR by increasing the FRET efficiency between the FPs and dyes. The system was used for four-color super-resolution imaging of target proteins and dynamic SIM tracking of mitochondria. In addition, the genic FRET fluorophore constructed with the ultra-stable mStayGold showed significant photobleaching resistance and can be used for long-term dynamic super-resolution imaging. Theoretically, by selecting appropriate FRET pairs, this method can improve the SNR of any fluorophore, providing a new labeling strategy for live-cell wash-free imaging.

biochemistry↗

bioRxiv2024

Reshaping epigenomic landscapes in facilitating the speciation of bread wheat

Polyploidization is a driving force of wheat evolution and speciation, yet its impact on epigenetic regulation and gene expression remains unclear. Here, we constructed a high-resolution epigenetic landscape across leaves, spikes, and roots of hexaploidy wheat and its tetraploid and diploid relatives. Inter-species stable-expression genes exhibited conserved amino acid sequences under strong purifying selection, while dynamic-expression genes were linked to species-specific adaptation. During hexaploidization, dominant D-subgenome homoeolog expression was suppressed via reduced activating epigenetic signals, converging expression with the A and B subgenomes. Proximal chromatin regions near genes were more stable, whereas distal regions, particularly enhancer-like elements mediated by H3K27ac and H3K4me3, exhibit higher dynamism. Sequence variations in these enhancers lead to differential gene regulation, influencing traits such as spike development. For instance, the two haplotypes of dCRE region of TaDEP-B1 resulted in significant differences in its expression and spikelet numbers. We also observed a coevolution of transcription factors and their binding sites, particularly within the expanded ERF family, which regulates spike morphology. This study highlights the interplay between sequence variation and epigenetic modifications in shaping transcriptional regulation during wheat speciation, offering valuable insights for genetic improvement.

plant biology↗

bioRxiv2024

Peptide Information Compression Technology (PICT): A New Frontier in Therapeutic Peptide Discovery

Therapeutic peptides hold tremendous market potential, yet the discovery methods for active peptides lag considerably behind those for small molecules and biological macromolecule drugs. Due to their respective limitations, traditional peptide compound synthesis and display libraries fail to meet the growing demands of the peptide drug market. Here, we introduce a novel Peptide Information Compression Technology (PICT) and, based on this technology, a large-scale physical peptide library and its accompanying high-throughput screening (HTS) platform. By screening for inhibitors of TSLP/TSLPR/IL7R and antagonists of MC3R at both the molecular and cellular levels, we have identified active molecules in the single-digital micromolar and nanomolar ranges, respectively. This demonstrates the immense application value of Peptide Information Compression Technology in the efficient discovery of peptide drugs.

molecular biology↗

bioRxiv2024

HCN1 channels in GABAergic amygdalar neurons underpin male-biased aggressive behaviors

Aggression behaviors typically vary between sexes, but the molecular mechanisms driving these disparities in neural coding are unclear. We found that aggression selectively activates GABAergic neurons in the posterior substantia innominata (pSI), an extend amygdala region critical for aggressive behaviors in both sexes of mice, with males exhibiting higher neuronal activity during the attack. Utilizing single-nucleus RNA sequencing, we characterized the diverse molecular landscape of pSI neurons, revealing significant differences in ion channels and hormone regulator genes that may underpin sex-specific aggression. Male GABAergic pSI neurons exhibited remarkable hyperexcitability due to increased Ih currents. Strikingly, modulating HCN1 expression not only adjusted this hyperexcitability but also influenced sexual dimorphism in aggression: silencing HCN1 in the GABAergic pSI neurons reduced male aggression, while its overexpression markedly heightened aggression in females. Furthermore, testosterone was shown to intensify aggression by upregulating HCN1 and remodeling pSI circuits. These findings provide detailed sex-specific molecular mechanisms underlying social behaviors.

neuroscience↗

bioRxiv2024

Cardiolipin-mimic lipid nanoparticles without antibody modification delivered senolytic in-vivo CAR-T therapy for inflamm-aging

mRNA-based in vivo CAR T cell engineering offers advantages over ex vivo therapies, including streamlined manufacturing and transient expression. However, current delivery requires antibody-modified vehicles with manufacturing challenges. In this study, inspired by cardiolipin, we identified a cardiolipin-like di-phosphoramide lipid that improved T cell transfection without targeting ligands, both in vivo and in vitro. The T cell-favored tropism is likely due to the lipids packing, shape, and rigidity. Encapsulating circular RNA further prolonged mRNA expression in the spleen and T cells. Using PL40 lipid nanoparticles, we delivered mRNA encoding a CAR targeting the senolytic and inflammatory antigen urokinase-type plasminogen activator receptor (uPAR), alleviating uPAR-related liver fibrosis and rheumatoid arthritis (RA). Single cell sequencing in humans confirmed uPARs relevance to senescence and inflammation in RA. To further enhance clinical translation, we screened and humanized scFvs against uPAR, establishing PL40 mRNA encoding a circular human uPAR CAR, with potential for treating aging-inflamed disorders. One Sentence SummaryWeve developed a unique class of Cardiolipin-mimic lipids that facilitate mRNA delivery to T cells in vivo without the need for antibody modification, enhancing the treatment of liver fibrosis and rheumatoid arthritis through circular CAR uPAR RNA and propelling the clinical application of humanized CAR against human uPAR.

bioengineering↗

bioRxiv2024

Estimating and correcting index hopping misassignments in single-cell RNA-seq data

BackgroundIndex hopping causes read assignment errors in data from multiplexed sequencing libraries. This issue has become more prevalent with the widespread use of high-capacity sequencers and highly multiplexed single-cell RNA sequencing (scRNA- seq). ResultsWe conducted deep, plate-based scRNA-seq on a mixed population of mouse skin cells. Analysis of transcriptomes from 1152 cells identified four distinct cell types. To estimate the error rate in sample assignment due to index hopping, we employed differential expression analysis to identify signature genes that were highly and specifically expressed in each cell type. We quantified the proportion of misassigned reads by examining the detection rates of signature genes in other cell types. Remarkably, regardless of gene expression levels, we estimated that 0.65% of reads per gene were assigned to incorrect cell across our data. To computationally compensate for index hopping, we developed a simple correction method wherein, for each gene, 0.65% of the librarys average expression level was subtracted from the expression in each cell. This correction had notable effects on transcriptome analyses, including increased cell-cell clustering distance and alterations in intermediate state assignments of cell differentiation. ConclusionsIndex hopping misassignments are measurable and can impact the experimental interpretation of sequencing results. We devised a straightforward method to estimate and correct for the index hopping rate by quantifying misassigned genes in distinct cell types within an scRNA-seq library. This approach can be applied to any barcoded, multiplexed scRNA-seq library containing cells with distinct expression profiles, allowing for correction of the expression matrix before conducting biological analysis.

genomics↗

bioRxiv2024

Inducible biosynthesis of bacterial cellulose in recombinant Enterobacter sp. FY-07

Bacterial cellulose (BC) is an extracellular polysaccharide with myriad unique properties, such as high purity, water-holding capacity and biocompatibility, making it attractive in materials science. However, genetic engineering techniques for BC-producing microorganisms are rare. Herein, the electroporation-based gene transformation and the {lambda} Red-mediated gene knockout method with a nearly 100% recombination efficiency were established in the fast-growing and BC hyperproducer Enterobacter sp. FY-07. This genetic manipulation toolkit was validated by inactivating the protein subunit BcsA in the cellulose synthase complex. Subsequently, the inducible BC-producing strains from glycerol were constructed through inducible expression of the key gene fbp in the gluconeogenesis pathway, which recovered more than 80% of the BC production. Finally, the BC properties analysis results indicated that the induced-synthesized BC pellicles were looser, more porous and reduced crystallinity, which could further broaden the application prospects of BC. To our best knowledge, this is the first attempt to construct the completely inducible BC-producing strains. Our work paves the way for increasing BC productivity by metabolic engineering and broadens the available fabrication methods for BC-based advanced functional materials.

synthetic biology↗

bioRxiv2024

Novel cell states arise in embryonic cells devoid of key reprogramming factors

The capacity for embryonic cells to differentiate relies on a large-scale reprogramming of the oocyte and sperm nucleus into a transient totipotent state. In zebrafish, this reprogramming step is achieved by the pioneer factors Nanog, Pou5f3, and Sox19b (NPS). Yet, it remains unclear whether cells lacking this reprogramming step are directed towards wild type states or towards novel developmental canals in the Waddington landscape of embryonic development. Here we investigate the developmental fate of embryonic cells mutant for NPS by analyzing their single-cell gene expression profiles. We find that cells lacking the first developmental reprogramming steps can acquire distinct cell states. These states are manifested by gene expression modules that result from a failure of nuclear reprogramming, the persistence of the maternal program, and the activation of somatic compensatory programs. As a result, most mutant cells follow new developmental canals and acquire new mixed cell states in development. In contrast, a group of mutant cells acquire primordial germ cell-like states, suggesting that NPS-dependent reprogramming is dispensable for these cell states. Together, these results demonstrate that developmental reprogramming after fertilization is required to differentiate most canonical developmental programs, and loss of the transient totipotent state canalizes embryonic cells into new developmental states in vivo.

developmental biology↗

bioRxiv2024

Molecular insights into the atypical activation mechanism of GPR156 in maintaining auditory function

The class C orphan GPCR GPR156, which lacks the typical extracellular region, plays a pivotal role in auditory function through Gi2/3. Here, we demonstrate that GPR156 with high constitutive activity is essential for maintaining auditory function, and we further present two cryo-EM structures of human GPR156. The GPR156 dimer in both the apo state and Gi3 protein-coupled state adopt a TM5/6-TM5/6 interface, indicating the high constitutive activity of GPR156 in the apo state. The C-terminus plays a dual role in promoting G protein binding within G-bound subunit while preventing the G-free subunit from binding to additional G protein. These observations explain how GPCR activity is maintained through dimerization and provide a mechanistic insight into the sustained role of GPR156 in maintaining auditory function.

molecular biology↗

bioRxiv2023

Genetically encoded affinity reagents (GEARs): A toolkit for visualizing and manipulating endogenous protein function in vivo.

Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional, universal, and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use nanobodies and single chain variable fragments (scFv), which recognize small epitopes, enabling fluorescent visualization and selective degradation of protein targets in vivo. Furthermore, we delineate a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad multifunctionality. We use GEARs to examine the native behaviour of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.

developmental biology↗

bioRxiv2023

Pan-genome analysis of different morphotypes reveals genomic basis of Brassica oleracea domestication and differential organogenesis

The domestication of Brassica oleracea has resulted in diverse morphological types with distinct patterns of organ development. Here we report a graph-based pan-genome of B. oleracea constructed with high-quality genome assemblies of different morphotypes. The pan-genome harbors over 200 structural variant (SV) hotspot regions enriched with auxin and flowering-related genes. Population genomic analyses reveal that early domestication of B. oleracea focused on leaf or stem selection. Gene flows resulting from agricultural practices and variety improvement are detected among different morphotypes. Selective sweep analysis identifies an auxin-responsive SAUR gene and a CLE family gene as the crucial players in the leaf-stem differentiation during the early stage of B. oleracea domestication, and the BoKAN1 gene as instrumental in shaping the leafy heads of cabbage and Brussels sprouts. Our pan-genome and functional analyses further discover that variations in the BoFLC2 gene play key roles in the divergence of vernalization and flowering characteristics among different morphotypes, and variations in the first intron of BoFLC3 are involved in fine-tuning the flowering process in cauliflower. This study provides a comprehensive understanding of the pan-genome of B. oleracea and sheds light on the domestication and differential organ development of this globally important crop species.

plant biology↗

bioRxiv2023

A comparative study of algorithms detecting differential rhythmicity in transcriptomic data

Rhythmic transcripts play pivotal roles in driving the daily oscillations of various biological processes. Genetic or environmental disruptions can lead to alterations in the rhythmicity of transcripts, ultimately impacting downstream circadian outputs, including metabolic processes and even behavior. To statistically compare the differences in transcript rhythms between two or more conditions, several algorithms have been developed to analyze circadian transcriptomic data, each with distinct features. In this study, we compared the performance of seven algorithms that were specifically designed to detect differential rhythmicity. We found that even when applying the same statistical threshold, these algorithms yielded varying numbers of differentially rhythmic transcripts. Nevertheless, the set of transcripts commonly identified as differentially rhythmic exhibited substantial overlap among algorithms. Furthermore, the phase and amplitude differences calculated by these algorithms displayed significant correlations. In summary, our study highlights a high degree of similarity in the results produced by these algorithms. Furthermore, when selecting an algorithm for analysis, it is crucial to ensure the compatibility of input data with the specific requirements of the chosen algorithm and to assess whether the algorithms output fits the needs of the user.

bioinformatics↗

bioRxiv2023

β1 integrins regulate cellular behaviors and cardiomyocyte organization during ventricular wall formation

AimsThe mechanisms regulating the cellular behavior and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. This study aims to determine whether and how {beta}1 integrins regulate cardiomyocyte behavior and organization during ventricular wall morphogenesis in the mouse. Methods and ResultsWe applied mRNA deep sequencing and immunostaining to determine the expression repertoires of /{beta} integrins and their ligands in the embryonic heart. Integrin {beta}1 subunit ({beta}1) and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, while fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1, which encodes the {beta}1 integrin subunit, was deleted via Nkx2.5Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts display an absence of trabecular zone but a thicker compact zone. The abundances of hyaluronic acid and versican are not significantly different. Instead, fibronectin, a ligand of {beta}1, was absent in B1KO. We examined cellular behaviors and organization via various tools. B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. The reduction of Notch1 activation was not the cause of the abnormal cellular organization in B1KO hearts. Mosaic clonal lineage tracing shows that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. Conclusions{beta}1 is asymmetrically localized in the cardiomyocytes, and its ECM ligands are enriched in the luminal side of the myocardium and surrounding cardiomyocytes. {beta}1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1, leading to ablation of {beta}1 integrins, causes the dissociation of cardiomyocytes from the ECM network and failure to establish tissue architecture to form trabeculae.

developmental biology↗